New therapeutic and diagnostic agents have begun to emerge from discovery efforts, which use high-throughput screening directed to certain gene-specific transcription factors.
One family of transcription factors responsible for transmitting a signal to a cell's nucleus are the proteins known as Signal Transducers and Activators of Transcription (STATs; see: Darnell et al. (1994) Science 264: 1415; for review, see: e.g., Ihle et al. (1994) Trends Biochem. Sci. 19: 222; Ihle et al. (1995) Trends Genetics 11: 69); and Horvath et al. (1997) Curr Opn Cell Biol. 9:233). STATs are activated by contact with the phosphorylated cytokine receptor; activation results in the STAT polypeptides forming a dimer and entering the nucleus, where the STAT dimer binds to the regulatory region of a gene that is inducible by the particular cytokine. Binding of the activated STAT dimer triggers transcription of the gene.
The STAT polypeptides (STAT1, STAT2, STAT4, STAT5a, STAT5b, and STAT6) have molecular masses from 84-113 kDa. Each STAT protein contains a Src homology-2 (SH2) domain capable of recognizing one or more phosphotyrosine sequences in the cytoplasmic portion of the activated receptor (see, Shuai et al. (1993) Nature 366: 580). Additionally, each cytokine receptor is specific for a particular STAT protein, and each STAT activates transcription of certain genes, thereby providing two layers of specificity in cytokine-induced signalling.
STAT6 and STAT4 are two proteins that are intimately involved in regulation of immune responses. STAT4 transduces to the nucleus signals from the IL-12 receptor. IL-12 is involved in the development of a TH1 immune response (see, Kaplan et al. (1996) Nature 382: 174-177), which is part of an organism's defense against intracellular pathogens. IL-12 is also necessary for the T-cell-independent induction of the cytokine interferon (IFN)-γ, which is a key step in the initial suppression of bacterial and parasitic infections. Knockout mice which lack STAT4 were found to be defective in all IL-12 functions tested, including the induction of IFN-gamma, mitogenesis, enhancement of natural killer cytolytic function and TH1 differentiation (see, Thierfelder et al. (1996) Nature 382: 171-174).
IL-4 signals are transduced to the nucleus by STAT6. In addition, IL-4 is a key cytokine in the initiation of a TH2 immune response, and also activates B and T lymphocytes. STAT6-deficient mice were shown to be deficient in IL-4 activities (see, Kaplan et al. (1996) Immunity 4: 313-319; Takeda et al. (1996) Nature 380: 627-630; Shimoda et al. (1996) Nature 380: 630-633).
Because of the importance of STAT4 and STAT6 in modulating the immune response of an organism, both in response to infection and in undesirable conditions such as inflammation, allergic reactions, and autoimmune diseases, a need exists by which the clinician can diagnose, enhance or reduce STAT4 and STAT6 signals. Intervention at the STAT level would have significant advantages compared to previous approaches, which typically target the IL-4 or IL-12 cytokine itself, or the interaction of the cytokine with the receptor. Disruption of cytokine function itself can cause a variety of undesirable side effects. These can be avoided by intervening at the level of STAT-mediated signal transduction.
WO 98/40478, published Sep. 17, 1998, discloses an oligonucleotide having 10 to 30 nucleotide units which is complementary to at least part of mRNA encoding human Stat-6 and is capable of inhibiting expression of Stat-6.
Identification of agents that can modulate STAT4 and STAT6-mediated signal transduction has heretofore been hampered by the lack of suitable assays. Recently, a new assay for identification of STAT6 and STAT4 signalling modulators was described (see, U.S. Pat. No. 6,207,391). Assay of binding of STAT4 and STAT6 to their corresponding receptors, and identification of agents which increase or decrease the degree of such binding, has now led to the identification of compounds which are useful in the diagnosis and treatment of various STAT-dependent conditions.
U.S. patent application Ser. No. 09/349,208, filed Jul. 7, 1999, and incorporated herein by reference, discloses STAT protein modulators useful for a variety of indications. Despite the advances made by the U.S. patent application Ser. No. 09/349,208, there remains a need for novel compounds capable of modulated STAT proteins such as STAT6. The present invention fulfills this and other needs.